There is societal need for new antibiotic compounds in our arsenal of defenses against bacterial pathogens, many of which are increasingly resistant to existing antibiotics. The best possible source for new antibiotic structures with potentially novel mechanisms of action is within natural environments, particularly soils, which have the greatest diversity of microbial life. This research proposal advances the science of metagenomics, the cloning of DNA from entire microbial communities, to discover novel antibiotics and identify the best lead candidates for clinical development. Scientists at the Lucigen Corporation and at Auburn University are uniting four key technological breakthroughs that together will result in the next generation of metagenomic libraries, a resource with greatly enhanced potential for antibiotic discovery. Specifically, the proposed research will identify antibiotic compounds using 1) an improved methodology for the isolation and purification of high molecular weight genomic DNA from soil microorganisms;2) a novel broad host range shuttle vector for enhanced expression of cloned DNAs;3) a random shear cloning method to produce very large insert sizes (>100 kb);and 4) a rapid and improved screening method to identify antibiotic-producing clones within a metagenomic library. The primary Phase I objectives are to produce the proof-of-concept next generation metagenomic library using the above technologies and to screen this library against bacterial and yeast tester strains to generate a collection of antibiotic- producing clones. Phase II will build upon the success of Phase I by constructing additional metagenomic libraries from multiple environmental samples, screening these libraries for antimicrobial activity, and, most importantly, characterizing the antimicrobial agents identified in Phase I and Phase II to determine the best lead candidates for clinical development. Lead candidates will have novel chemical structures, have high potency against multiple bacterial pathogens (e.g., MRSA), and minimal toxicity for eukaryotic cells. Each of the different technologies necessary for the proposed research has been proven effective separately;therefore, the synthesis of these different methods has a high probability of success and also represents a significant advancement for the science of antibiotic discovery. Furthermore, the libraries produced from this research are a valuable genomic resource that may be screened for other bioactive compounds (e.g., with anticancer or antiviral activities) in subsequent research. PUBLIC HEALTH RELEVANCE: The use of antibiotics to treat bacterial disease has been a success story in the history of modern medicine, and yet there is still a need to identify new antibiotics that can treat bacterial infections, particularly ones caused by multi-drug resistant pathogens. This research will combine four different technological breakthroughs to enable antibiotic discovery from microorganisms in natural environments (e.g., soils) by harvesting and expressing their genetic pathways directly, without the need to cultivate the different microorganisms in a laboratory. In this way, this technology will access the antibiotics produced by a great diversity of microorganisms, many of which are unknown to science, and will identify the best novel antibiotic compounds for use in treating bacterial disease.